Most human pre-mRNA transcripts are alternatively spliced, but the significance and fine-tuning of alternative splicing in different biological processes is only starting to be understood. SRSF3 (SRp20) is a member of a highly conserved family of splicing factors that have critical roles in key biological processes, including tumor progression. Here, we show that SRSF3 regulates cellular senescence, a p53-mediated process to suppress tumorigenesis, through TP53 alternative splicing. Downregulation of SRSF3 was observed in normal human fibroblasts undergoing replicative senescence, and was associated with the upregulation of p53 beta, an alternatively spliced isoform of p53 that promotes p53-mediated senescence. Knockdown of SRSF3 by short interfering RNA (siRNA) in early-passage fibroblasts induced senescence, which was associated with elevated expression of p53 beta at mRNA and protein levels. Knockdown of p53 partially rescued SRSF3-knockdown-induced senescence, suggesting that SRSF3 acts on p53-mediated cellular senescence. RNA pulldown assays demonstrated that SRSF3 binds to an alternatively spliced exon uniquely included in p53 beta mRNA through the consensus SRSF3-binding sequences. RNA crosslinking and immunoprecipitation assays (CLIP) also showed that SRSF3 in vivo binds to endogenous p53 pre-mRNA at the region containing the p53 beta-unique exon. Splicing assays using a transfected TP53 minigene in combination with siRNA knockdown of SRSF3 showed that SRSF3 functions to inhibit the inclusion of the p53 beta-unique exon in splicing of p53 pre-mRNA. These data suggest that downregulation of SRSF3 represents an endogenous mechanism for cellular senescence that directly regulates the TP53 alternative splicing to generate p53 beta. This study uncovers the role for general splicing machinery in tumorigenesis, and suggests that SRSF3 is a direct regulator of p53.Oncogene advance online publication, 9 July 2012; doi:10.1038/onc.2012.288.Genotoxic stressors, such as radiation, induce cellular damage that activates pre-programmed repair pathways, some of which involve microRNAs (miRNA) that alter gene expression. The let-7 family of miRNA regulates multiple cellular processes including cell division and DNA repair pathways. However, the role and mechanism underlying regulation of let-7 genes in response to stress have yet to be elucidated. In collaboration with Nicole Simone et al., we demonstrate that let-7a and let-7b expression decreases significantly following exposure to agents that induce stress including ionizing radiation. This decrease in expression is dependent on p53 and ATM in vitro and is not observed in a p53(-/-) colon cancer cell line (HCT116) or ATM(-/-) human fibroblasts. Chromatin Immunoprecipitation (ChIP) analysis showed p53 binding to a region upstream of the let-7 gene following radiation exposure. Luciferase transient transfections demonstrated that this p53 binding site is necessary for radiation-induced decreases in let-7 expression. A radiation-induced decrease in let-7a and let-7b expression is also observed in radiation-sensitive tissues in vivo and correlates with altered expression of proteins in p53-regulated pro-apoptotic signaling pathways. In contrast, this decreased expression is not observed in p53 knock-out mice suggesting that p53 directly repress let-7 expression. Exogenous expression of let-7a and let-7b increased radiation-induced cytotoxicity in HCT116 p53(+/+) cells but not HCT116 p53(-/-) cells. These results are the first demonstration of a mechanistic connection between the radiation-induced stress response and the regulation of miRNA and radiation-induced cytotoxicity and suggest that this process may be a molecular target for anticancer agents.